Tissue damage caused by the presence of cytokines and growth factors during severe inflammatory conditions (a.k.a. runaway inflammatory conditions) can result from blockage of blood flow due to stroke, myocardial infarction (ischemia) or surgical intervention, infectious diseases, parasitic and other inflammatory conditions. It is believed that cytokines are responsible for "communication" between cells which ultimately leads to gene activation. These discrete signaling molecules are considered benign to tissue in a healthy state. Cytokines include interleukins, various growth factors, interferons, and colony-stimulating factors. However, the net biological effect of the interaction of this class of molecules can also be inflammation and in this instance, cytokines are also known to play a major role in a wide variety of disease states such as cancer, allergy, infection, inflammation, angiogenesis, and differentiation. Cytokines and growth factors together are also believed to play a major role in restenosis (neointimal hyperplasia or proliferation following percutaneous transluminal coronary angioplasty and related procedures for removing blockages within blood vessels and lymph ducts).
The mechanism by which tissue is damage during a runaway inflammatory response caused, for example, by a bacterial infection is as follows: Inflammatory cytokines such as tumor necrosis factor and interleukin-1 activate intracellular, microbiocidal neutrophils. In their normal, defensive operation, neutrophils aggregate and release toxic granule proteins and the products of neutrophil oxidatively burst to destroy the harmful microbe. However, in a runaway process, the neutrophil aggregation and release of microbiocidal substances are not localized in the vicinity of the microbe, leading to damage of the host tissue. This chain of events occurs in a number of diseases associated with, e.g., infectious agents of the immune system, chronic inflammation, and the respiratory system, as well as during numerous surgical procedures.
Accordingly, it is a goal of the invention to use thalidomide to ameliorate tissue damaged by severe inflammatory responses, without interfering with host tissue defense mechanisms. In particular, the invention recognizes the great need for ameliorative drug therapies for stroke, heart attack, restenosis, adult respiratory distress syndrome (ARDS) and septic shock (the latter two conditions having a high likelihood of fatality), asthma, hearing loss associated with bacterial meningitis, inflammatory bowel conditions, and dozens of other conditions.
Blood flow reductions in the heart can result in dysfunction of this organ and cell death if the flow reduction is severe enough. Restoration of coronary blood flow early during a heart attack is becoming a clinical reality with the advent and improvements in thrombolytic, mechanical, and surgical interventions. While early restoration of blood flow by thrombolysis or following transient ischemia can prevent or mitigate the degree of cell death (infarction), reperfusion can still result in some degree of cardiac dysfunction or cell death (also referred to as stunned myocardia). Thus, it would be of great clinical value to find a means to preserve normal function of the heart during reperfusion and during various forms of cardiac surgery.
Oxygen free radicals and oxygen intermediates, especially singlet oxygen and the hydroxyl radical, cause extensive tissue damage. Free radicals and oxygen intermediates within living cells arise from endogenous sources, for example, from mitochondrial electron transport chain, oxidant enzymes, phagocytic cells and auto-oxidation reactions, as well as from exogenous sources such as cigarette smoke, "redox cycling" drugs and pesticides, heat stress, and ionizing radiation. These oxygen species damage compounds of all biochemical classes, including nucleic acids, protein and free amino acids, lipids and lipoproteins, carbohydrates, and connective tissue macromolecules. In addition, these oxygen species are believed to have an impact on cellular activities, such as membrane function, metabolism, and gene expression. Both singlet oxygen and hydroxyl radical are known to produce damaging strand breaks in DNA during reperfusion (specifically, during the reoxygenation aspect of reperfusion following ischemia) and during severe inflammatory conditions. Also, these oxidants, in the presence of metal ions (e.g., iron), initiate lipid peroxidation, which, in turn, produces mutagens, carcinogens, and other reactive oxygen species.
Numerous clinical conditions implicate oxygen radicals as the cause of tissue damage in single organs such as erythrocytes (e.g., lead poisoning), lungs (e.g., acute respiratory distress syndrome), heart and cardiovascular system (e.g., atherosclerosis), kidney (aminoglycoside nephrotoxicity), GI tract (e.g., free-fatty-acid-induced pancreatitis), brain and CNS (e.g., senile dementia, hypertensive cerebrovascular injury, and cerebral trauma), eye (e.g., cataractogenesis), and skin (e.g., solar radiation and contact dermatitis). Clinical conditions involving multiorgan disorders linked to oxygen radicals include, for example, inflammatory-immune injury, ischemia/reperfusion states, radiation injury, aging, cancer and amyloid diseases.
In spite of the more recently known deleterious effects of these oxygen species, the scientific community has focused its attention on the superoxide anion for the past two decades, leaving largely unexplored the role of singlet oxygen and the hydroxyl radical in human disease. Thus, no drugs that specifically target these oxygen species are currently available. It is one of the goals of the present invention to use a series of drugs that can inhibit the "runaway" inflammatory response in tissue experiencing oxidative stress. In particular, the need for such drugs is particularly great for protecting or treating cardiac tissue undergoing heart attack, angioplasty, and cardiac surgery. The invention also addresses the need for ameliorative drug therapies for ischemic stroke, vasospasm during subarachnoid hemorrhage, head injury, spinal cord injury, and neurosurgery, whereby the effects of damage from reperfusion and inflammation would be prevented or lessened.
Additionally, heart disease is the biggest cause of death in the Western world. There are many different forms of heart disease and disease states that can develop from a number of different factors including stress, diet, tobacco use, and genetic make up of the individual. Ischemia is a heart disease condition characterized as a local hypoxia caused by mechanical obstruction or occlusion of the blood supply. Oxygen radicals have been implicated as important mediators of tissue injury during myocardial ischemia and reperfusion. A number of studies have shown that free radicals, particularly superoxide anions (O.sub.2.sup.-) and hydroxyl radicals are generated following reperfusion of the ischemic myocardium and have linked the free radical generation to the loss of contractile function. Superoxide artion is relatively unreactive and is considered dangerous because its dismutation results in the formation of hydrogen peroxide which can potentially generate the highly reactive hydroxyl radical (OH) in the presence of transition metal ions. It is therefore generally believed that ultimate tissue damage occurs due to OH radicals. Indirect proof for the involvement of OH radicals in ischemia/reperfusion injury is derived from observations of a protective effect of OH radical scavengers such as dimethylthiourea (DMTU), dimethylsulfoxide, and mannitol. In addition, certain agents which prevent the formation of hydroxyl radicals have also demonstrated a protective effect, including deferoxamine, superoxide dismutase, and catalase.
Another active oxygen species is singlet molecular oxygen (.sup.1 O.sub.2). Singlet oxygen is not a radical; rather, it is an electronically excited state of oxygen which results from the promotion of an electron to higher energy orbitals. In Kukreja et al., Biochim. Biophys. A, 990:198-205 (1990); the Kukreja et al., Am. J. Physiol., 259:H1330-H1336 (1989), data was presented which demonstrated that superoxide anion or hydrogen peroxide are the least reactive species in damaging sarcolemma or sarcoplasmic reticulum. Therefore, it might be inferred that the only species believed to be injurious in myocardial tissue is the OH radical that can initiate lipid peroxidation which can in turn, produce lipid free radicals that may become important sources of singlet oxygen in vivo. Hence, the damage often attributed to the OH radical could be the resultant effects of other intermediate reaction products including lipid free radicals and singlet oxygen.
Janero et al., J. Mol. Cell Cardiol., 21:1111-1124 (1989), showed that alpha-tocopherol provides cellular protection by acting as a chain breaker in the lipid peroxidation process, not by scavenging the O.sub.2 --radical per se. Singlet oxygen is also acted upon by alpha-tocopherol. Hearse et al., Circ. Res., 65:146-153 (1989), and Vandeplassche et al., J. Mol. Cell Cardiol., 22:287-301 (1990) (abstract) showed that .sup.1 O.sub.2 generated from exogenous sources is able to mimic ischemia/reperfusion-induced myocardial damage. Tarr et al., J. Mol. Cell Cardiol., 21:539-543 (1989), recently reported that rose bengal, when applied extracellularly to frog atrial myocytes, induced a prolongation followed by a reduction of action potential duration. In addition, Donck et al., J. Mol. Cell Cardiol., 20:811-823 (1988) reported that isolate myocytes exposed to rose bengal light rapidly experience ultrastructural injury.
In Kukreja et al., Abs. of 63rd Sci. Sess. (AHA) (Dallas), 1068 (1990), it was reported that singlet oxygen generated from photosensitization of rose bengal induced significant inhibition of calcium uptake and Ca.sup.2+ -ATPase activity in isolate sarcoplasmic reticulum. This damage caused by singlet oxygen could be significantly reduced using L-histidine, but not with SOD or catalase. Misra et al., J. Biol. Chem., 265:15371-15374 (1990), reported that L-histidine is a scavenger of singlet oxygen. In contrast, SOD and catalase are scavengers of superoxide anion. Kim et al., Am. J. Physiol., 252:H252-H257 (1987), demonstrated that L-histidine provides significant protection of sarcolemmal Na.sup.+ K.sup.+ -ATPase activity following ischemia/reperfusion in guinea pig hearts.
As explained above, ischemia is a decreased blood supply to a body part or organ which is often marked by pain and organ dysfunction. Ischemic tissues become hypoxic and in some extreme cases anoxic. Pathologic processes due to ischemia such as heart disease and strokes are the first and third leading causes of death in the United States respectively. However, a substantial proportion of tissue injury that accompanies ischemia is not directly due to the lack of oxygen getting to tissues but rather occurs during reperfusion which is when oxygen is reintroduced into the tissues (McCord, N. E. J. M., 312:159-163, 1985). During reperfusion there is a conversion of oxygen into superoxide and secondarily-derived oxygen radicals which trigger a series of events that culminate in massive tissue damage (Granger et al., Covan. Physiol. Bhaum, 71:67-75, 1981; Roy et al., In; Greenwald, R., Cohen. G. Eds., Oxyradicals and their Scavenger systems, Vol. 2., Cellular and Molecular Aspects, New York; Elsevier Science, 1983; pages 154-157; Parks, Gastroenterology, 82:9-15, 1982). The pathway by which superoxide is produced is catalyzed by the enzyme xanthine oxidase which is derived during ischemia from xanthine dehydrogenase. Xanthine dehydrogenase is widely distributed among tissues. It converts rapidly to xanthine oxidase as a result for proteolysis and sulfhydryl oxidation (Batelli et al., Biochem. J., 126:747-749, 1972; Della Corte, Biochem. J. 126:739-745, 1972). There appears to be a correlation between xanthine dehydrogenase levels in tissue and their susceptibility to ischemia/reperfusion injury, with organs such as the intestines, heart and lungs that have high xanthine dehydrogenase levels being more susceptible to injury while skeletal muscle which has low levels of xanthine dehydrogenase being relatively resistant to such injury (McCord, N. E. J. M., 312:159-163 (1985).
McCord, N. E. J. M., 312:159-163 (1985), hypothesized that xanthine dehydrogenase is converted to xanthine oxidase when blood flow to a tissue is decreased to the point where availability of oxygen limits ATP production. This results in a drop in cellular energy levels and the Ca.sup.2+ ion gradient across cellular membranes can no longer be maintained. An elevated cytosolic Ca.sup.2+ concentration occurs which in turn activates a protease that converts xanthine dehydrogenase to xanthine oxidase. Concurrently, depletion of cellular ATP during ischemia results in an elevated AMP concentration. Such AMP is catabolized to hypoxanthine which upon reperfusion, reacts with oxygen in a chemical reaction catalyzed by the newly formed xanthine oxidase yielding oxygen radicals as an end product.
It should be emphasized at this point that even though generation of superoxide and other oxygen-derived radicals lead to tissue damage, reperfusion of ischemic tissues itself results in very little damage (Korthuis et al., Am. J. Physiol., 256:11315, 1989; Perry et al., Am. J. Physiol., 254:6366, 1988). Zimmerman and Granger, Surg. Clin. North America, 72:(1)65-83 (1992) determined that these oxidants mediate microvascular permeability, which develops after one hour of reperfusion, and lesions are produced by three hours during ischemia/reperfusion. They hypothesized that xanthine oxidase-derived oxidants were produced in epithelial and endothelial cells. Such oxidants mediate production and release of pro-inflammatory cytokines which attract, activate and promote adherence of neutrophils to microvascular endothelium. These neutrophils are believed to be the primary mediator of reperfusion injury (Granger, Amer. J. Physiol., 255:H 1269-H 1275, 1988). A number of studies using in vitro models of ischemia/reperfusion injury have supported this contention. For example, exposure of endothelial cells to anoxia-reoxygenation resulted in increased intracellular levels of xanthine oxidase, increased generation of oxygen derived radicals, cell dysfunction and death. Inhibitors of xanthine oxidase prevented production of oxygen derived radicals and cellular damage (Zweier et al., Proc. Natl. Acad. Sci. (U.S.A.), 85:4045-4050, 1988; Ratych et al., Surgery, 102:122-131, 1987; Inauen et al., Free Radical Biol. Med., 9:219-23, 1990b). Neutrophils added to anoxic and reperfused endothelial cells adhered significantly to these cells and this adherence was markedly reduced by superoxide dismutase which suggested that the superoxide radical mediates anoxia/reperfusion-induced neutrophil adhesion to endothelial cells (Suzuki et al., Am. J. Physiol., 257:H1740-1745, 1989; Yoshida et al., Am. J. Physiol. 262:H1891-H1898, 1992). Oxygen radicals such as superoxide stimulate endothelial cells to produce inflammatory cytokines such as platelet activating factor (PAF) and leukotriene B.sub.4 (LTB.sub.4) (Granger and Kvietys, Can. J. Physiol. Pharmaceutical, 71:67-75, 1993; Zimmerman and Granger, Surg. Clin. North Amer., 72:(1)65-83, 1992). Mangino et al., Amer. J. Physiol., 257:6299 (1989) detected a 687% increase in mucosal LTB.sub.4 levels in the dog ileum model during reperfusion, but no increase was initially seen during ischemia. Zimmerman et al., Gastroenterology, 99:1358-1363 (1990) saw a 200% increase in LTB.sub.4 after reperfusion of the cat intestine. This group further demonstrated that reperfusion-induced neutrophil infiltration was significantly reduced in animals pretreated with lipooxygenase inhibitor or a LTB.sub.4 receptor antagonist which indicates that LTB.sub.4 mediates reperfusion-induced neutrophil infiltration into the surrounding tissue. It has also been demonstrated that a PAF receptor antagonist and catalase ameliorated the adhesion-promoting ability of supernatants from reperfused endothelial cells (Yoshida et al., Amer. J. Physiol., 262:H1891-H1898, 1992). A PAF receptor antagonist L659, 989 also was shown to prevent hypoxia-induced neutrophil adhesion to human umbilical vein endothelial cells (Milhoan et al., Amer. J. Physiol., 256:H956-H962, 1992). DuBois et al., J. Immunol., 143:964-70 (1989) reported that PAF mediates TNF-alpha generation in alveolar macrophages through endogenous LTB.sub.4 production. TNF-alpha, in turn, is capable of inducing release of large amounts of PAF, as seen in rat peritoneal macrophage leading DuBois et al. to hypothesized that LTB.sub.4 or PAF antagonists may prove to be effective therapeutics to block reperfusion-induced inflammation.
Another pro-inflammatory cytokine, IL-1 beta, has been demonstrated to play a causal role in ischemia/reperfusion injury of the canine gracilis muscle (Ascer, Amer. Vas. Surg., 6:69-73, 1992) and in cerebral ischemia in rats (Minami et al., J. Neurol., 58:390-392, 1992). Neutrophil accumulation seen in rat heart ischemia/reperfusion injury was shown to be IL-1 beta-dependent (Brown et al., Proc. Natl. Acad. Sci. (U.S.A.), 87:5926-30, 1990). Similar to TNF-alpha, IL-1 beta production was enhanced by LTB.sub.4 (Rola-Pleszczynski and Lemaire, J. Immunol, 135:3958, 1985). TNF-alpha and IL-1 beta have been shown to be involved in neutrophil-mediated inflammatory damage at a number of levels. TNF-alpha and IL-1 beta produced by macrophages induces significant increases in E-selectin receptor expression on endothelial cells within two hours. Such E-selectin expression enhanced the ability of endothelial cells to initially trap and bind neutrophils at a body site where injury is occurring. By 24 hours, however, expression of E-selectin on endothelial cells begins to wane and is replaced by increased expression of ICAM-1 which is also stimulated by TNF-alpha and IL-1 beta (Lipsky, Springer Semin. Immunopathol, 11:123-162, 1989). The neutrophils initially bound to E-selectin then bind to ICAM-1. Such binding of neutrophils to ICAM-1 is an important step towards diapedesis of neutrophils which must happen before neutrophil-mediated reperfusion injury can occur. TNF-alpha also upregulates CD 11/CD 18 receptors on neutrophils which are responsible for binding to the previously described ICAM-1 endothelial cell receptors (Gamble et al., Proc. Natl. Acad. Sci. (U.S.A.), 82:8667, 1985).
Seekamp et al., Amer. J. Pathol., 143:(2)453-63 (1993) showed that in a rat hind-limb ischemia model where TNF-alpha and IL-1 beta plasma levels increased significantly within one hour of reperfusion, polyclonal antibody to TNF-alpha and IL-1 beta conferred significant protection from vascular injury in both lung and muscle. Similar results were also obtained using soluble TNF-alpha receptor and IL-1 receptor antagonist. E-selectin expression in lung vasculature was blocked when animals were treated with anti-TNF-alpha antibodies. This work indicates that therapeutic strategies which inhibit TNF-alpha and IL1 beta production may be effective in limiting reperfusion-based tissue damage. It has already been demonstrated that thalidomide inhibits TNF-alpha production in erythema nodosum leprosum patients (Sarno et al., Clin. Exp. Immunol, 84:103-108, 1991) and in vitro in monocytes (Sampaio et al., J. Exp. Med., 173:699-703, 1991) while Shannon et al., American Society Microbiology Annual Meeting-Abstract U-53 (1990) indicated that thalidomide inhibited IL-1 beta production in vitro. It is the purpose of this invention, therefore, to use thalidomide to inhibit ischemia/reperfusion-based inflammatory damage from occurring by administering thalidomide alone or in combination with other anti-ischemia/reperfusion and antiinflammatory therapeutics including, but not limited, to other inhibitors of cytokines such as TNF-alpha, IL-1 beta, PAF and/or LTB.sub.4 Such other current anti-ischemia/reperfusion injury therapeutics include: